CN104854124A - Macrocyclic ketoamide immunoproteasome inhibitors - Google Patents

Abstract

The present invention relates to compounds of formula (I) and pharmaceutically acceptable salts thereof, wherein X, Y, Z, R ¹ , R ² , R ³ and R ^3' are as defined in the detailed description of the invention and the claims. In addition, the present invention relates to methods of making and using compounds of formula I as well as pharmaceutical compositions containing such compounds. Compounds of formula I are LMP7 inhibitors useful in the treatment of related inflammatory diseases and conditions such as rheumatoid arthritis, lupus and irritable bowel disease.

Description

Macrocyclic ketamide immunoproteasome inhibitors

field of invention

The present invention relates to organic compounds useful in the treatment and/or prevention of inflammatory diseases or conditions in mammals, in particular to macrocyclic ketoamide compounds useful in the treatment of rheumatoid arthritis, lupus and irritable bowel disease (IBD) , their preparation, pharmaceutical compositions containing them and their use as LMP7 inhibitors.

Background of the invention

LMP7 is a major component of the immunoproteasome and is mainly expressed in immune cells such as T/B lymphocytes and monocytes, as well as in non-immune cells that have been exposed to inflammatory cytokines, including IFN-γ and TNFα. The immunoproteasome plays a fundamental role in the production of antigenic peptide repertoires and in shaping MHC class I-restricted CD8+ T cell responses. Moebius J. et al., European Journal of Immunology. 2010; Basler, M. et al. Journal of Immunology. 2004.3925-34. Emerging data suggest that, in addition to modulating MHC class I-mediated antigen presentation, LMP7 also regulates inflammatory cytokine production and immune cell function.

A small molecule LMP7 inhibitor - PR-957 - has been shown to potently block Th1/17 differentiation, B cell effector functions and inflammatory cytokines (IL-6, TNF-α, IL-23) produce. Muchamuel T. et al., Natural Medicine. 2009.15, 781-787; Basler M. et al., Journal of Immunology. 2010, 634-41.

Additionally, blocking LMP7 with PR-957 has been shown to yield therapeutic benefit in several preclinical autoimmune disease models. First, PR-957 was shown to significantly reduce disease scores in mouse CAIA and CIA arthritis models, with significantly reduced markers of inflammation and bone erosion. Muchamuel T. et al., Natural Medicine. 2009.15, 781-787. In addition, PR-957 reduced plasma cell numbers and anti-dsDNA IgG levels in the MRL/lpr lupus-prone mouse model and prevented disease progression in these mice. Ichikawa HT et al., Arthritis & Rheumatism. 2012. 64, 493-503. Furthermore, PR-957 reduced inflammation and tissue destruction in a mouse model of DSS-induced colitis. Basler M. et al., Journal of Immunology. 2010, 634-41. Finally, in an IBD model, LMP7 knockout mice have also been shown to be disease-free. Schmidt N. et al., Gut 2010. 896-906.

In conclusion, the data highly suggest that LMP7 activity is closely related to B/T lymphocyte function and inflammatory cytokine production, all of which are clinically proven targets/pathways in the pathogenesis of rheumatoid arthritis, lupus and IBD . Thus, the existing data already provide a strong rationale for targeting LMP7 for autoimmune disease indications. Due to the potential propensity for chronic use of covalent inhibitors in chronic diseases such as autoimmunity, covalently reversible or non-covalent small molecule LMP7 inhibitors are highly desirable for autoimmune disease indications.

Summary of the invention

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

in:

X is _CH2 , O or NH;

Y is CH or N;

Z is CH or N;

R ¹ is C _1-7 alkyl, C _3-8 cycloalkyl or -CH ₂ -phenyl;

R ² is C _1-7 alkyl or -CH ₂ -phenyl; and

One of R ³ or R ^3′ is hydrogen and the other is C _3-8 cycloalkyl, unsubstituted C _1-7 alkyl or C _1-7 alkyl substituted by C _1-7 alkoxy; or

R ³ and R ^3' are taken together with the carbon atom to which they are attached to form a C _3-8 cycloalkyl moiety.

The invention also provides pharmaceutical compositions comprising the compounds, methods of using the compounds and methods of making the compounds.

All documents cited or relied upon are expressly incorporated herein by reference.

Detailed description of the invention

Unless otherwise indicated, the following specific terms and phrases used in the specification and claims are defined as follows:

The term "moiety" refers to an atom or group of chemically bonded atoms that is bonded to another atom or molecule by one or more chemical bonds, thereby forming part of a molecule. For example, the variable R of formula I refers to a moiety bound to the core structure of formula I by a covalent bond.

For a particular moiety having one or more hydrogen atoms, the term "substituted" refers to the fact that at least one hydrogen atom of the moiety is replaced by another substituent or moiety. For example, the term "C _1-7 alkyl substituted by halogen" refers to the fact that one or more hydrogen atoms of a C _1-7 alkyl (as defined below) are replaced by one or more halogen atoms (e.g. trifluoromethane group, difluoromethyl, fluoromethyl, chloromethyl, etc.).

The term "alkyl" refers to an aliphatic straight or branched chain saturated hydrocarbon moiety having 1-20 carbon atoms. In certain embodiments, the alkyl group has 1-10 carbon atoms.

The term "C _1-7 alkyl" refers to an alkyl moiety having 1-7 carbon atoms. Examples of C _1-7 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

The term "C _1-7 alkoxy" refers to a group of formula -O-R' wherein R' is alkyl. Examples of C _1-7 alkoxy moieties include methoxy, ethoxy, isopropoxy and tert-butoxy.

"Aryl" refers to a monovalent cyclic aromatic hydrocarbon moiety having a mono-, bi- or tricyclic aromatic ring. Aryl groups may be optionally substituted as defined herein. Aryl moieties include, but are not limited to, phenyl, naphthyl, phenanthrenyl, fluorenyl, indenyl, pentalenyl, azulenyl, oxydiphenyl, biphenyl, methylenediphenyl , aminodiphenyl, diphenylsulfinyl (diphenylsulfidyl), diphenylsulfonyl, diphenylisopropylidene, benzodioxanyl, benzofuranyl, benzodioxine benzodioxylyl, benzopyranyl, benzoxazinyl, benzoxazinone, benzopiperadinyl, benzopiperazinyl, benzopyrrolidinyl, benzomorpholinyl , methylenedioxyphenyl, ethylenedioxyphenyl, etc., including their partially hydrogenated derivatives, are each optionally substituted.

The terms "halo", "halogen" and "halide" are used interchangeably to refer to fluorine, chlorine, bromine or iodine.

"C _3-8 cycloalkyl" refers to a monovalent saturated carbocyclic moiety having a monocyclic or bicyclic ring. The C _3-8 cycloalkyl moiety may be optionally substituted with one or more substituents. Examples of C _3-8 cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, including partially unsaturated (cycloalkenyl) derivatives thereof.

Unless otherwise indicated, the term "hydrogen" refers to a hydrogen atom moiety (-H), rather than _H2 .

Unless otherwise indicated, the term "compound of formula" refers to any compound selected from the class of compounds defined by said formula (including any pharmaceutically acceptable salt or ester of any such compound, unless otherwise indicated).

The term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free base or free acid, which are neither biologically nor otherwise undesirable. Salts can be prepared with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., preferably hydrochloric acid and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, salicylic acid, Succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, N-acetyl cysteine, etc. Alternatively, salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts, and the like. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, Salts of amines, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins, etc.

The compounds of the present invention may exist in the form of pharmaceutically acceptable salts. The compounds of the invention may also exist in the form of pharmaceutically acceptable esters, ie methyl and ethyl esters of acids of formula I intended for use as prodrugs. The compounds of the invention may also be solvated, ie hydrated. Solvation may take place during production, or may occur, ie as a result of the hygroscopic properties (hydration) of the initially anhydrous compound of formula I.

Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space are termed "isomers" and are within the scope of the present invention. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Diastereomers are stereoisomers that have relative configurations at one or more chiral centers, which are not enantiomers. Stereoisomers that bear one or more asymmetric centers that are non-superimposable mirror images of each other are termed "enantiomers". When a compound has an asymmetric center, for example if a carbon atom is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of one or more of their asymmetric centers and described by the R- and S-sequence rules of Cahn, Ingold and Prelog, or by the way molecules rotate the plane of polarized light , and assigned as dextrorotatory or levorotatory (ie as (+) or (-)-isomer, respectively). Chiral compounds may exist as individual enantiomers or as mixtures thereof. A mixture containing equal amounts of the enantiomers is termed a "racemic mixture".

The term "therapeutically effective amount" of a compound refers to the amount of the compound effective to prevent, alleviate or ameliorate disease symptoms or prolong the survival time of the treated subject. Determination of a therapeutically effective amount is within the skill of the art. The therapeutically effective amount or dosage of a compound of the invention may vary within wide limits and may be determined by methods known in the art. In each particular case, such dosages will be adjusted according to the individual requirements, including the particular compound administered, the route of administration, the condition being treated, and the patient being treated. Generally, when administered orally or parenterally to an adult human weighing about 70 Kg, a daily dosage of about 0.1 mg to about 5,000 mg, 1 mg to about 1,000 mg, or 1 mg to 100 mg may be appropriate, although it may be exceeded when indicated. upper and lower bounds. The daily dose can be administered as a single dose or in multiple doses, or for parenteral administration it can be given as a continuous infusion.

The term "pharmaceutically acceptable carrier" is intended to include any and all substances compatible with pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other substances compatible with pharmaceutical administration. substances and compounds. Unless any conventional media or materials are incompatible with the active compounds, their use in the compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutically acceptable carriers that can be used to prepare the compositions can be solid, liquid or gaseous; thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enteric coatings or other protected formulations (i. resin or encapsulated in lipid-protein vesicles), sustained-release preparations, solutions, suspensions, elixirs, aerosols, and the like. The carrier can be selected from a variety of oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose and glycols are preferred liquid carriers, especially for injectable solutions (when isotonic with blood). For example, formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient prepared by dissolving the solid active ingredient in water to produce an aqueous solution and sterilizing the solution. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glyceryl monostearate Esters, Sodium Chloride, Skimmed Milk Powder, Glycerin, Propylene Glycol, Water, Ethanol, etc. The composition may receive conventional pharmaceutical additives such as preservatives, stabilizers, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like. Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E.W. Martin. In any case, such compositions contain an effective amount of the active compound together with a suitable carrier to prepare a suitable dosage form for suitability to the recipient.

In the practice of the method of the present invention, an effective amount of any one compound of the present invention or any one compound of the present invention or its pharmaceutically acceptable amount is administered via any conventional and acceptable method known in the art alone or in combination. Use a combination of salts or esters. Thus, a compound or composition may be administered orally (e.g., buccally), sublingually, parenterally (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppository or lotion), transdermally (e.g., by electroporation of the skin). ) or by inhalation (for example by aerosol) and in the form of solid, liquid or gas doses, including tablets and suppositories. Administration can be in single dose form with continuous treatment or in single dose treatment ad libitum. Therapeutic compositions may also be in the form of oil emulsions or dispersions in combination with lipophilic salts such as pamoic acid or in the form of biodegradable sustained release compositions for subcutaneous or intramuscular administration.

In detail, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

in:

X is _CH2 , O or NH;

Y is CH or N;

Z is CH or N;

R ¹ is C _1-7 alkyl, C _3-8 cycloalkyl or -CH ₂ -phenyl;

R ² is C _1-7 alkyl or -CH ₂ -phenyl; and

One of R ³ or R ^3′ is hydrogen and the other is C _3-8 cycloalkyl, unsubstituted C _1-7 alkyl or C _1-7 alkyl substituted by C _1-7 alkoxy; or

R ³ and R ^3' together with the carbon atom to which they are attached form a C _3-8 cycloalkyl moiety.

In another embodiment, the invention provides compounds of formula (I), wherein X is _CH2 .

In another embodiment, the invention provides compounds of formula (I), wherein X is O or NH.

In another embodiment, the invention provides compounds of formula (I), wherein Y is CH.

In another embodiment, the invention provides compounds of formula (I), wherein Y is N.

In another embodiment, the invention provides compounds of formula (I), wherein Z is CH.

In another embodiment, the invention provides compounds of formula (I), wherein Z is N.

In another embodiment, the invention provides compounds of formula (I), wherein X is _CH2 , Y and Z are CH.

In another embodiment, the invention provides compounds of formula (I), wherein R ¹ is methyl, -CH ₂ -phenyl or cyclopropyl.

In another embodiment, the invention provides compounds of formula ( ^I ), wherein R1 is _-CH2 -phenyl.

In another embodiment, the invention provides compounds of formula (I), wherein R2 is butyl or ^-CH2 _- phenyl.

In another embodiment, the invention provides compounds of formula (I), wherein R2 is ^-CH2 _- phenyl.

In another embodiment, the invention provides compounds of formula (I), wherein one of ^R3 or _R3 ^' is hydrogen and the other is cyclopropyl, methyl or _-CH2OCH3 .

In another embodiment, the invention provides compounds of formula (I), wherein ^R3 is methyl and R3 ^' is hydrogen.

In another embodiment, the invention provides compounds of formula (I), wherein ^R3 and R3 ^' are taken together to form a cyclopropyl moiety together with the carbon atom to which they are attached.

A particular embodiment of the invention relates to compounds of formula (I'):

wherein R ¹ , R ² and R ³ are as defined above; more particularly R ¹ is -CH ₂ -phenyl, R ² is -CH ₂ -phenyl and R ³ is methyl.

In another embodiment, the present invention provides a compound of formula (I), wherein said compound is:

In another embodiment, the present invention provides a compound of formula (I), wherein said compound is:

In another embodiment, the present invention provides a compound of formula (I), wherein said compound is (9S,12S)-12-methyl-11,14-dioxo-10,13-diaza- ^Tricyclo [15.3.1.12,6]doco-1(20),2(22),3,5,17(21),18-hexene-9-carboxylic acid ((S)-1-benzyl Base-2-benzylcarbamoyl-2-oxoethyl)-amide.

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides compounds of formula (I) for use as therapeutically active substances.

In another embodiment, the present invention provides the use of a compound of formula (I) for the treatment or prevention of inflammatory diseases or disorders, in particular selected from rheumatoid arthritis, lupus and intestinal Irritable illness.

In another embodiment, the present invention provides the use of a compound of formula (I) in the preparation of a medicament for the treatment or prevention of an inflammatory disease or disorder, which is particularly selected from the group consisting of rheumatoid arthritis inflammation, lupus and irritable bowel disease.

In another embodiment, the present invention provides a compound of formula (I) for use in the treatment or prevention of an inflammatory disease or disorder, particularly selected from the group consisting of rheumatoid arthritis, lupus and irritable bowel disease provoking disease.

In another embodiment, the present invention provides a method of treating an inflammatory disease or condition selected from rheumatoid arthritis, lupus, and irritable bowel disease (IBD), the method comprising administering to a subject in need thereof the treatment an effective amount of a compound of formula (I).

In another embodiment, there is provided the invention as described above.

The starting materials and reagents used in the preparation of these compounds are generally available from commercial suppliers such as Aldrich Chemicals Co. or can be prepared by methods known to those skilled in the art as given in documents such as: Fieser and Fieser's Reagents for Organic Synthesis; Wiley & Sons: New York, 1991, Vols 1-15; Rodd's Chemistry of Carbon Compounds, Elsevier Scientific Publishing Company, 1989, Vols 1-5 and Supplements; and Organic Reactions, Wiley & Sons: New York, 1991, Vols 1 - 40 rolls.

The following synthetic reaction schemes illustrate only some of the ways in which the compounds of the present invention may be synthesized. Various modifications to these synthetic reaction schemes are possible and will be suggested to those skilled in the art having reference to the disclosure contained herein.

The starting materials and intermediates of the synthetic reaction schemes can be isolated and purified, if desired, using conventional techniques including, but not limited to, filtration, distillation, crystallization, chromatography, and the like. Such materials can be characterized using conventional means, including physical constants and spectral data.

Unless indicated to the contrary, the reactions described herein are preferably carried out under an inert atmosphere at atmospheric pressure at about -78°C to about 150°C, more preferably at about 0°C to about 125°C, most preferably and conveniently at about room temperature ( or ambient temperature) such as at a temperature of about 20°C.

The compounds of the invention can be prepared in any number of conventional ways. For example, they can be prepared as outlined in Schemes 1-4 below.

Process 1

As shown in Scheme 1, N-Boc protected amino acid 1 can be converted to Weinreb amide 2, which can then be reduced to aldehyde 3 using lithium aluminum hydride (LiAlH ₄ ). The aldehyde can be treated immediately with acetone cyanohydrin to form the new cyanohydrin 4 as a mixture of diastereomers. The nitrile can be hydrolyzed to formic acid and the Boc protecting group lost by treatment with hydrochloric acid. The Boc group can be reintroduced using di-tert-butylpyrocarbonate to give acid 5, which can then be coupled with an appropriate amine 6 using a reactive reagent such as HATU to afford hydroxyamide 7.

Process 2

As shown in Scheme 2, acid 8 can be coupled with an appropriately selected amino acid 9 to give amide 10 using an activating reagent such as HATU.

Process 3

As shown in Scheme 3, bis(pinacolato)diboron) 12 can selectively borylate appropriately protected homophenylalanine derivatives 11 under an iridium catalyst (similar to Org. The method described in Lett. 2010, 12, 3870), yielding 13 as the major regioisomer. Biaryl derivative 14 can be prepared by Suzuki coupling of 10 and 13. Biaryl derivatives 14 can then be deprotected with trifluoroacetic acid (TFA) to give amino acids 15. Compound 16 can be obtained by macrocyclization under highly dilute conditions in the presence of an activating reagent such as HATU. The key acid intermediate 17 can be obtained by hydrolysis of the ester using trimethyltin hydroxide (Angew. Chem. Int. Ed. 2005, 44, 1378).

Process 4

As shown in Scheme 4, hydroxyamide 7 can be treated with TFA. The resulting free amine salt can be coupled in situ with acid 17 using an activating reagent such as HATU to afford hydroxyamide 18. Ketoamide 19 can be provided by oxidation with Dess-Martin periodinane.

Example

While a number of illustrative embodiments are depicted and described herein, compounds of the present invention can be prepared using appropriate starting materials, according to methods outlined herein, and/or by methods available to those skilled in the art. All reactions involving air-sensitive reagents were performed under an inert atmosphere. Reagents were used as obtained from commercial suppliers unless otherwise indicated.

Example 1

(9S,12S)-12-methyl-11,14-dioxo-10,13-diaza-tricyclo[15.3.1.1 ^2,6 ]doco-1(20),2(22 ),3,5,17(21),18-hexene-9-carboxylic acid ((S)-1-benzyl-2-benzylcarbamoyl-2-oxo-ethyl)-amide

step 1

To a solution of (S)-2-tert-butoxycarbonylamino-3-phenyl-propionic acid (25 g, 94.34 mmol) in DMF (250 mL) was added N,O-dimethyl Hydroxylamine hydrochloride (13.72 g, 141.50 mmol), HATU (37.64 g, 99.05 mmol) and N,N-diisopropylethylamine (50.70 mL, 283.01 mmol). The reaction mixture was stirred at room temperature for 16 hours, then diluted with ethyl acetate (1000 mL) and washed with water (5 x 250 mL). The organic layer was dried and concentrated under reduced pressure. The crude residue was purified by CombiFlash column chromatography with 20% EtOAc in hexanes to afford 27.5 g (94%) of (S)-1-(methoxy-methyl-carbamoyl)-2-phenyl -Ethyl]-tert-butyl carbamate as a colorless oil. LC/MS: (M+H) ⁺ = 309.0.

step 2

Add (S)-1-(methoxy-methyl-carbamoyl)-2-phenyl-ethyl]-carbamic acid tert-butyl ester (15g, 48.70mmol) in THF (180mL) at 0°C To the stirred solution of LiAlH ₄ (1.0 M solution in THF, 57 mL, 57 mmol) was added. The reaction mixture was stirred at 0 °C for 1 h, then carefully quenched by portionwise addition of sodium sulfate decahydrate until gas evolution ceased. EtOAc was added and the reaction mixture was stirred vigorously at room temperature for 30 minutes, then filtered. The filtrate was dried and concentrated under reduced pressure to afford 11.0 g (91%) of ((S)-1-benzyl-2-oxo-ethyl)-carbamic acid tert-butyl ester as a white solid without Used after further purification.

step 3

To a solution of ((S)-1-benzyl-2-oxo-ethyl)-carbamic acid tert-butyl ester (7.0 g, 28.1 mmol) in DCM (80 mL) was added acetone cyanohydrin ( 7.16 g, 84.3 mmol) and triethylamine (2.36 mL, 16.86 mmol). The reaction was stirred at room temperature for 3 hours, then water was added and the organics were removed under reduced pressure. The aqueous residue was extracted with ethyl acetate and washed twice with water. The organic layer was dried and concentrated under reduced pressure. The crude residue was purified by CombiFlash column chromatography using 20% EtOAc in hexanes as mobile to afford 5.0 g (58%) of ((S)-1-benzyl-2-cyano-2-hydroxy- Ethyl)-tert-butyl carbamate, yellow oil. LC/MS: (M+H) ⁺ =277.4.

step 4

A solution of ((S)-1-benzyl-2-cyano-2-hydroxy-ethyl)-carbamic acid tert-butyl ester (5.0 g, 18.11 mmol) in 6M HCl (90 mL) was heated at 100 °C 16 hours, then cooled to room temperature and concentrated in vacuo to afford 4.0 g (95%) of (S)-3-amino-2-hydroxy-4-phenyl-butyric acid hydrochloride as a grayish-yellow solid without Used after further purification. LC/MS: (M+H) ⁺ = 196.2.

step 5

To a solution of (S)-3-amino-2-hydroxy-4-phenyl-butyric acid hydrochloride (18.0g, 77.9mmol) in 1,4-dioxane (150mL) and water (150mL) Sodium bicarbonate (65.45 g 779 mmol) and di-tert-butyl dicarbonate (25.48 g, 116.9 mmol) were added. The mixture was stirred vigorously at room temperature for 16 hours. The organic phase was removed under reduced pressure. The remaining heterogeneous aqueous layer was diluted with water (200 mL), extracted with Et2O ( ₂ x 200 mL, discarded). The aqueous layer was adjusted to pH = 3 by addition of 2M aqueous HCl, extracted with EtOAc (3 x 400 mL). The combined extracts were dried and concentrated under reduced pressure to afford 18.0 g (78%) of (S)-3-tert-butoxycarbonylamino-2-hydroxy-4-phenyl-butanoic acid as an off-white solid. LC/MS: (M+H) ⁺ = 296.6.

step 6

To a stirred solution of (S)-3-tert-butoxycarbonylamino-2-hydroxy-4-phenyl-butanoic acid (10.0 g, 33.89 mmol) in DMF (150 mL) was added benzylamine (4.35 g, 40.67mmol), HATU (14.16g, 37.28mmol) and N,N-diisopropylethylamine (6.56g, 50.84mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 3 hours, then diluted with ethyl acetate (800 mL), washed with ice-cold water (2 x 950 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The crude residue was purified by CombiFlash column chromatography using EtOAc in 30% hexanes to afford 7.3 g (56%) of (S)-1-benzyl-2-benzylcarbamoyl-2-hydroxy-ethyl )-tert-butyl carbamate as a white solid. LC/MS: (M+H) ⁺ = 385.2.

step 7

Add 3-(3-bromophenyl)propionic acid (800mg, 3.49mmol), (S)-2-alanine tert-butyl hydrochloride (698mg, 3.84mmol) and HATU (1.46g, To a solution of 3.84 mmol) in DMF (8 mL) was added N,N-diisopropylethylamine (1.83 mL, 10.5 mmol). The yellow reaction mixture was stirred at room temperature overnight, then quenched with water, extracted with EtOAc (2x). The combined organics were washed with water (3x) and brine, then dried over _MgSO4 and concentrated. The residue was purified by chromatography (10% to 30% EtOAc/hexanes) to afford 1.11 g (89%) of (S)-2-[3-(3-bromo-phenyl)-propionylamino]-propane Acid tert-butyl ester, viscous colorless oil. ¹ H NMR (300MHz, CDCl ₃ ) δ: 7.30-7.38(m, 2H), 7.10-7.21(m, 2H), 5.99(d, J=6.8Hz, 1H), 4.46(quin, J=7.1Hz, 1H), 2.86-3.04(m, 2H), 2.36-2.62(m, 2H), 1.46(s, 9H), 1.32(d, J=7.2Hz, 3H).

Step 8

To a solution of (S)-2-(tert-butoxycarbonylamino)-4-phenylbutanoic acid (1.0 g, 3.58 mmol) in MeOH (20 mL) was added dropwise trimethylsilyl heavyweight at 0 °C. Nitrogen methane (2.0M in _Et2O , 3.6 mL, 7.2 mmol). Additional trimethylsilyldiazomethane (2.0 M in _Et2O ) was added in 1 mL aliquots until a pale yellow color persisted. A total of 9 mL (-5 eq) of reagent was added. The reaction was quenched with a few drops of acetic acid, at which point the solution became colorless. The mixture was concentrated. The residue was taken up on silica gel and purified by chromatography (10% to 30% EtOAc/hexanes) to afford 1.03 g (98%) of (S)-2-tert-butoxycarbonylamino-4-phenyl-butane Acid methyl ester, a colorless oil. ¹ H NMR (300MHz, CDCl ₃ )δ: 7.27-7.33(m, 2H), 7.15-7.24(m, 3H), 5.07(d, J=7.9Hz, 1H), 4.31-4.44(m, 1H), 3.73 (s, 3H), 2.64-2.73 (m, 2H), 2.09-2.25 (m, 1H), 1.88-2.03 (m, 1H), 1.46 (s, 9H).

step 9

In a 35 mL pressure tube place pinacol diborate (952 mg, 3.75 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (37 mg, 0.14 mmol) and [Ir(OMe)COD )] ₂ (45 mg, 0.07 mmol). A solution of (S)-methyl 2-tert-butoxycarbonylamino-4-phenyl-butyrate (1.0 g, 3.41 mmol) in hexane (16 mL) was added. The tube was purged with nitrogen, then sealed and heated at 65°C for 16 hours. _The dark red reaction was cooled to room temperature, diluted with _CH2Cl2 , transferred to a flask and concentrated. The residue was taken up on silica gel and purified by chromatography (10% to 25% EtOAc/hexanes) to isolate 900 mg of a viscous colorless oil. NMR analysis indicated an approximately 3:1:1 mixture of (S)-2-tert-butoxycarbonylamino-4-[3-(4,4,5,5-tetramethyl-[1,3 ,2]dioxaborolan-2-yl)-phenyl]-butyric acid methyl ester: ¹ H NMR (300MHz, CDCl ₃ ) δ: 7.66 (d, J=6.9Hz, 1H), 7.64(s,1H),7.31-7.33(m,1H),7.21(d,J=8.1Hz,1H),5.08(d,J=7.6Hz,1H),4.38(br.s.,1H), 3.75(s,3H),2.65-2.74(m,2H),2.11-2.23(m,1H),1.89-2.02(m,1H),1.48(s,9H),1.37(s,12H);(S )-2-tert-butoxycarbonylamino-4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)- Phenyl]-butyric acid methyl ester: ¹ H NMR (300MHz, CDCl ₃ )δ: 7.75(d, J=7.8Hz, 2H), 7.31-7.35(m, 2H), 5.08(d, J=7.6Hz ,1H),4.38(br.s.,1H),3.75(s,3H),2.65-2.74(m,2H),2.11-2.23(m,1H),1.89-2.02(m,1H),1.48( s,9H), 1.36(s,12H); and (S)-4-[3,5-bis-(4,4,5,5-tetramethyl-[1,3,2]dioxaborin Heterocyclopentane-2-yl)-phenyl]-2-tert-butoxycarbonylamino-butyric acid methyl ester: ¹ H NMR (300MHz, CDCl ₃ )δ:8.14(s,1H),7.73(s ,2H),5.08(d,J=7.6Hz,1H),4.38(br.s.,1H),3.74(s,3H),2.65-2.74(m,2H),2.11-2.23(m,1H) ,1.89-2.02(m,1H),1.47(s,9H),1.36(s,24H).

Step 10

To (S)-2-[3-(3-bromo-phenyl)-propionylamino]-propionic acid tert-butyl ester (918mg, 2.58mmol) and (S)-2-tert-butoxycarbonylamino- 4-[3-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-butyric acid methyl ester (from The main component of the mixture of step 3, 900 mg, 2.15 mmol) in 1,4-dioxane (12 mL) was added Pd (PPh ₃ ) ₄ (124 mg, 0.11 mmol) and 2.0 M aqueous sodium carbonate (3.2 mL, 6.4mmol). The biphasic mixture was stirred at 90 °C for 3.5 hours, then cooled to room temperature, quenched with water, extracted with EtOAc (2x). The combined organics were dried over _MgSO4 and concentrated. The residue was taken up on silica gel and purified by chromatography (20% to 40% EtOAc/hexanes) to isolate 647 mg (53%) of a viscous colorless oil. NMR analysis indicated (S)-2-tert-butoxycarbonylamino-4-{3'-[2-((S)-1-tert-butoxycarbonyl-ethylcarbamoyl)-ethyl]- Biphenyl-3-yl}-butyric acid methyl ester and (S)-2-tert-butoxycarbonylamino-4-{3'-[2-((S)-1-tert-butoxycarbonyl- Ethylcarbamoyl)-ethyl]-biphenyl-4-yl}-butyric acid methyl ester about 3:1 mixture. LC/MS: (M+Na) ⁺ = 591.

step 11

To (S)-2-tert-butoxycarbonylamino-4-{3'-[2-((S)-1-tert-butoxycarbonyl-ethylcarbamoyl)-ethyl]-biphenyl To a solution of -3-yl}-butyric acid methyl ester (major component of the mixture from step 4, 647 mg, 1.14 mmol) in dichloromethane (8 mL) was added TFA (2 mL, 26.0 mmol). The pale yellow reaction mixture was stirred at room temperature for 2.5 hours. Additional TFA (2 mL) was added, stirring was continued for 2.5 hours, then the mixture was concentrated and chased with dichloromethane (3x). The residue was dissolved in dichloromethane (200 mL) and DMF (50 mL). HATU (519 mg, 1.37 mmol) was then added followed by N,N-diisopropylethylamine (1.6 mL, 9.1 mmol). The yellow reaction mixture was stirred at room temperature for 96 hours. Dichloromethane was removed under reduced pressure. Water (200 mL) was added while cooling with ice. The aqueous layer was extracted with _Et2O (200 mL), then EtOAc (200 mL). The combined organics were washed with water (4x) and brine, then dried over _MgSO4 and concentrated. The crude residue was taken up on silica gel and purified by chromatography first with 50% to 100% EtOAc/hexanes, then with 0% to ₂ % MeOH/ _CH2Cl2 to afford 83 mg (19%) of (9S, 12S)-12-methyl-11,14-dioxo-10,13-diaza-tricyclo[15.3.1.1 ^2,6 ]doco-1(20),2(22),3 ,5,17(21),18-Hexene-9-carboxylic acid methyl ester as a white solid. LC/MS: (M+H) ⁺ = 395; ¹ H NMR (300MHz, CDCl ₃ ) δ: 7.27-7.47 (m, 5H), 7.04-7.11 (m, 3H), 6.64 (d, J = 7.9Hz ,1H),6.40(d,J=8.3Hz,1H),4.86-5.00(m,1H),4.18(ddd,J=11.4,7.6,4.0Hz,1H),3.68(s,3H),3.20( ddd, J＝13.8, 10.8, 2.6Hz, 1H), 2.86(ddd, J＝13.8, 8.5, 2.6Hz, 1H), 2.66(dd, J＝8.5, 4.5Hz, 2H), 2.55-2.63(m, 1H), 2.34-2.46(m, 1H), 2.15-2.30(m, 1H), 1.94-2.08(m, 1H), 1.40(d, J=6.8Hz, 3H).

Step 12

To (9S,12S)-12-methyl-11,14-dioxo-10,13-diaza-tricyclo[15.3.1.1 ^2,6 ]dococarbon-1(20),2( 22), 3,5,17(21), a solution of methyl 18-hexene-9-carboxylate (80mg, 0.20mmol) in 1,2-dichloroethane (3mL) was added trimethylhydrogen Tin oxide (183 mg, 1.01 mmol). The cloudy reaction mixture was stirred at 80 °C for 6 hours, then cooled to room temperature and concentrated. The residue was partitioned between EtOAc and 1.0M HCl. The aqueous layer was extracted with EtOAc. The combined organics were washed with 1.0M HCl (5x), dried over MgSO ₄ , then concentrated and dried under high vacuum to give (9S,12S)-12-methyl-11,14-dioxo-10,13 -Diaza-tricyclo[15.3.1.1 ^2,6 ]doco-1(20),2(22),3,5,17(21),18-hexene-9-carboxylic acid, white Semi-solid which was used without further purification.

Step 13

To a solution of ((S)-1-benzyl-2-benzylcarbamoyl-2-hydroxy-ethyl)-carbamic acid tert-butyl ester (93 mg, 0.24 mmol) in dichloromethane (2 mL) TFA (0.50 mL, 6.5 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, then concentrated and chased with dichloromethane (2x), dried under high vacuum to give a pale yellow oil. To this oil was added (9S,12S)-12-methyl-11,14-dioxo-10,13-diaza-tricyclo[15.3.1.1 ^2,6 ]doco-1 (20 ), 2(22), 3,5, 17(21), 18-hexene-9-carboxylic acid (crude from step 6, 77 mg, 0.20 mmol) in DMF (2 mL). Then HATU (85 mg, 0.22 mmol) and N,N-diisopropylethylamine (0.18 mL, 1.01 mmol) were added. The yellow reaction mixture was stirred overnight at room temperature, then quenched with water. The resulting precipitate was collected by filtration, washed with water, dried by suction and then dried under high vacuum to yield 110 mg (84%) of (9S,12S)-12-methyl-11,14-dioxo-10,13-di Aza-tricyclo[15.3.1.1 ^2,6 ]doco-1(20),2(22),3,5,17(21),18-hexene-9-carboxylic acid ((S)- 1-Benzyl-2-benzylcarbamoyl-2-hydroxy-ethyl)-amide as an off-white solid and mixture of epimers. LC/MS: (M+Na) ⁺ = 669.

Step 14

To (9S,12S)-12-methyl-11,14-dioxo-10,13-diaza-tricyclo[15.3.1.1 ^2,6 ]dococarbon-1(20),2( 22), 3,5,17(21), 18-hexene-9-carboxylic acid ((S)-1-benzyl-2-benzylcarbamoyl-2-hydroxyl-ethyl)-amide (105mg, To a slightly cloudy solution of 0.16 mmol) in dichloromethane (8 mL) was added Dess-Martin periodinane (103 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 2 hours, during which time a thick precipitate had formed. Sat. NaHCO ₃ (5 mL) and 10% Na ₂ S ₂ O ₃ (5 mL) were added to quench. The biphasic mixture was stirred vigorously for 20 minutes, then the layers were separated. The aqueous layer was extracted with dichloromethane (2x). The combined organic layers were washed with saturated NaHCO ₃ , then dried over MgSO ₄ and concentrated to a pale yellow solid. Trituration with MeOH/ _Et2O gave 38 mg (36%) of (9S,12S)-12-methyl-11,14-dioxo-10,13-diaza-tricyclo[15.3.1.1 ^2,6 ] Docos-1(20), 2(22), 3,5,17(21), 18-hexene-9-carboxylic acid ((S)-1-benzyl-2-benzylcarbamoyl -2-Oxo-ethyl)-amide as a white solid. LC/MS: (M+Na) ⁺ = 667; ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 9.20 (t, J = 6.4Hz, 1H), 8.52 (d, J = 8.1Hz, 1H), 8.19(d, J=9.1Hz, 1H), 8.01(d, J=6.8Hz, 1H), 7.16-7.52(m, 14H), 7.08-7.15(m, 4H), 5.12-5.19(m, 1H) ,4.70-4.81(m,1H),4.31(d,J=6.4Hz,2H),3.79-3.88(m,1H),3.02-3.09(m,1H),3.00(dd,J=9.3,6.1Hz ,1H),2.78-2.88(m,2H),2.75(d,J=6.3Hz,2H),2.53-2.59(m,1H),2.15(ddd,J=12.9,9.5,2.9Hz,1H), 1.83-1.92 (m, 2H), 1.16 (d, J=7.1Hz, 3H).

Example 2

The following compounds were prepared by procedures similar to those described in Example 1:

Example 3

Analysis protocol and results

Cell-Based Proteasome Activity/Selectivity Assays

The cell-based proteasome subunit activity/selectivity assay is a set of 5 fluorescent assays that independently measure the activity of β5c or β5i (chymotrypsin-like activity), β2c associated with the proteasome complex in cultured cells /2i (trypsin-like) and β1c or β1i (caspase-like) protease activity. Specifically, the following substrates were used for each subunit activity: β1i:(PAL) _2Rh110 ; β1c:(LLE) _2Rh110 ; β2c/2i:(KQL) _2Rh110 ; β5c:(WLA) _2Rh110 ; β5i : (ANW) ₂ Rh110. The following actions were followed:

Cell preparation: 25 μl of Ramos cells (2×10 ⁶ /ml in DPBS) were plated in half area plates (PerkinElmer Cat 6005569) to a final 5× ^{10 4} cells/well. Add 0.5 [mu]l of 100x 4-fold serial dilutions of test compound or DMSO to each well. The highest compound concentration tested was 20 μM, so serial dilutions of compounds started at 200 mM. Incubate at 37°C for 30 minutes. Then equilibrate at room temperature for 15 minutes. 25 μl of a 2x reaction mixture consisting of 0.025% digitonin, 20 μM of each substrate and 0.5M sucrose in DPBS was added. Shake at 700 rpm for 1 minute. Incubate for 120 minutes at room temperature. Plates were then read using an Envision multi-label plate reader (PerkinElmer) at 500 nm excitation/519 nm emission.

Proteasome Activity Analysis of Modified PBMC

This cell-based proteasome activity assay is similar to the previous Ramos cell-based assay in terms of substrates, but employs human PBMCs in complete RPMI with 10% FBS as reaction buffer. This assay was designed to evaluate the level of cell penetration of test compounds in primary human cells. The following protocol was followed: Freshly isolated PBMCs from healthy donors were plated at 1x105 cells/well in 100 μl complete RPMI with ¹⁰ % FBS in V-bottom 96-well plates. Add 1 [mu]l of 100X 4-fold serially diluted compound/well and incubate for 1 hour. The highest compound concentration tested was 20 [mu]M (100X working stock, starting at 2 mM). The cells were spun down at 2000 rpm for 5 minutes. Remove all supernatant. Cells were then resuspended in 25 [mu]l DPBS and transferred to fresh half area plates (PerkinElmer Cat 6005569). In a final reaction in a volume of 50 μl consisting of 25 μl cell suspension, 0.5 μl 100x inhibitor or DMSO, 25 μl substrate mix (containing 0.025% digitonin, 20 uM substrate (substrate: (PAL) _Rh110 , (LLE) ₂ Rh110, (KQL) ₂ Rh110, (WLA) ₂ Rh110 or (ANW) ₂ Rh110)/in a mixture of 10% FBS and 0.5M sucrose). Shake for 1 minute (at 700 rpm). After 2 hours of incubation, the plate was read using an Envision plate reader at 500 nm excitation/519 nm emission.

PBMC IP-10 Analysis

PBMCs were isolated from whole blood as follows: Blood was collected in heparinized tubes in a sterile environment. Blood was diluted with an equal volume of PBS/2% FCS and 30ml of this mixture was added to ACCUSPIN tubes containing 15ml Histopaque-1077 which had been centrifuged at 800g for 30 seconds and warmed to room temperature. The tubes were then centrifuged at 800 g for 20 minutes at room temperature without abrupt braking. The monomolecular bands just above the polyethylene glaze were removed by a Pasteur pipette. These monomolecular cells were washed three times with sterile PBS, counted, and resuspended to approximately ^1.5x106 /ml in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 1 mM pyruvate Sodium, penicillin (50U/ml), streptomycin (50μg/ml) and glutamine (2mM). Approximately ^2x105 cells/well were plated into 96-well tissue culture plates (BD Falcon 353072) and pre-incubated with increasing doses of compound in a final concentration of 1% DMSO for 60 mi/37°C. Cells were then stimulated with CpG Type A (Invivogen, Cat#tlrl-2216; ODN 2216) at a final concentration of 2.5 μΜ. Cells were grown overnight and the supernatant was removed. Cells remaining in the wells were assayed for PBMC viability using the ATPlite Luminescence Assay (Perkin-Elmer) following the manufacturer's instructions. Luminescence was measured on a Perkin-Elmer Envision using luminescence filters. IP10 levels were determined using the CXCL10/IP10 AlphaLISA kit (Perkin-Elmer) following the manufacturer's instructions except that all volumes were halved. Luminescence was measured on an Envision Multilabel plate reader using AlphaScreen standard settings.

result :

Table 1 below presents the results of the above assays for representative compounds of the invention, where IC50 and EC50 activity values are given in μM:

Table 1

It is to be understood that the present invention is not limited to the particular embodiments of the invention described above, since modifications may be made to the particular embodiments and still fall within the scope of the appended claims.

Claims (23)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: X is _CH2 , O or NH; Y is CH or N; Z is CH or N; R ¹ is C _1-7 alkyl, C _3-8 cycloalkyl or -CH ₂ -phenyl; R ² is C _1-7 alkyl or -CH ₂ -phenyl; and One of R ³ or R ^3′ is hydrogen and the other is C _3-8 cycloalkyl, unsubstituted C _1-7 alkyl or C _1-7 alkyl substituted by C _1-7 alkoxy; or R ³ and R ^3' are taken together with the carbon atom to which they are attached to form a C _3-8 cycloalkyl moiety. 2. A compound according to claim 1, wherein X is _CH2 . 3. The compound according to claim 1, wherein X is O or NH. 4. The compound according to claim 1, wherein Y is CH. 5. The compound according to claim 1, wherein Y is N. 6. The compound according to claim 1, wherein Z is CH. 7. The compound according to claim 1, wherein Z is N. 8. The compound according to claim 1, wherein X is _CH2 , Y and Z are CH. 9. The compound according to claim ¹ , wherein R1 is methyl, _-CH2 -phenyl or cyclopropyl. 10. The compound according to claim ¹ , wherein R1 is _-CH2 -phenyl. 11. The compound according to claim 1, wherein R2 is butyl or ^-CH2 _- phenyl. 12. The compound according to claim 1, wherein one of ^R3 or _R3 ^' is hydrogen and the other is cyclopropyl, methyl or _-CH2OCH3 . 13. The compound according to claim 1, wherein ^R3 is methyl and R3 ^' is hydrogen. 14. The compound according to claim 1, wherein ^R3 and R3 ^' are taken together with the carbon atom to which they are attached to form a cyclopropyl moiety. 15. The compound according to claim 1, wherein said compound is: 16. The compound according to claim 1, wherein said compound is: 17. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-16 and a pharmaceutically acceptable carrier. 18. Compounds according to any one of claims 1 to 16 for use as therapeutically active substances. 19. Use of a compound according to any one of claims 1-16 for the treatment or prevention of an inflammatory disease or condition selected from rheumatoid arthritis, lupus and irritable bowel disease. 20. Use of a compound according to any one of claims 1-16 for the preparation of a medicament for the treatment or prevention of an inflammatory disease or disorder selected from the group consisting of rheumatoid arthritis, lupus and irritable bowel cause disease. 21. A compound according to any one of claims 1-16 for use in the treatment or prevention of an inflammatory disease or condition selected from rheumatoid arthritis, lupus and irritable bowel disease. 22. A method of treating an inflammatory disease or condition selected from rheumatoid arthritis, lupus and irritable bowel disease, the method comprising administering a therapeutically effective amount of a compound according to any one of claims 1-16 to a subject in need thereof A step of. 23. The invention as hereinbefore described.